Anti-adhesive barrier membrane using alginate and hyaluronic acid for biomedical applications

ABSTRACT

A non-synthetic, hydrophilic, biodegradable, biocompatible polysaccharide based non-toxic anti-adhesion hydrogel barrier is disclosed herein. The barrier of the present invention is formed by constructing a unique interpenetrating, crosslinked network with a unique porosity. Furthermore, the barrier of the present invention is comprised of tunable biopolymers for controllable mechanical robustness and degradation. The barrier of the present invention effectively reduces unwanted adhesions using non-synthetic components.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/596,685, filed May 16, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/803,258 filed on Jul. 20, 2015, now U.S. Pat.No. 9,656,001, issued May 23, 2017, which is a continuation of U.S.patent application Ser. No. 13/269,344, filed on Oct. 7, 2011, now U.S.Pat. No. 9,095,558, issued on Aug. 4, 2015, which claims priority toU.S. Provisional Patent Application Ser. No. 61/391,299, filed on Oct.8, 2010. The content of each of the above applications is herebyincorporated by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant no.BES0201744 and BES0500969 awarded by the National Science Foundation.The government has certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of biopolymers,and more particularly to a non-toxic, anti-adhesion hydrogel barriercomprising biocompatible polysaccharides.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

None.

REFERENCE TO A SEQUENCE LISTING

None.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with the porous biopolymer hydrogels and methods ofpreparing the same.

WIPO Patent Publication No. WO 2009/108760 A8 (Zawko and Schmidt, 2009)discloses a hydrogel and a method of making a porous hydrogel bypreparing an aqueous mixture of an uncrosslinked polymer and acrystallizable molecule; casting the mixture into a vessel; allowing thecast mixture to dry to form an amorphous hydrogel film; seeding the castmixture with a seed crystal of the crystallizable molecule; growing thecrystallizable molecule into a crystal structure within theuncrosslinked polymer; crosslinking the polymer around the crystalstructure under conditions in which the crystal structure within thecrosslinked polymer is maintained; and dissolving the crystals withinthe crosslinked polymer to form the porous hydrogel.

U.S. Patent Publication No. 20100209509 (Kao et al., 2010) discloseshydrogels wherein a polymer matrix is modified to contain a bifunctionalpoly(alkylene glycol) molecule covalently bonded to the polymer matrix.The hydrogels can be cross-linked using, for example, glutaraldehyde.The hydrogels may also be crosslinked via an interpenetrating network ofa photopolymerizable acrylates. The hydrogels may also be modified tohave pharmacologically-active agents covalently bonded to thepoly(alkylene glycol) molecules or entrained within the hydrogel. Livingcells may also be entrained within the hydrogels.

SUMMARY OF THE INVENTION

The present invention discloses a non-toxic anti-adhesion hydrogelbarrier, composed of non-synthetic, hydrophilic, biodegradable,biocompatible polysaccharides formed by constructing a uniqueinterpenetrating, crosslinked network with a unique porosity. Theinvention further describes a method for preparing the same.

In one embodiment the instant invention provides a method of making aporous anti-adhesion hydrogel comprising the steps of: (i) preparing anaqueous mixture of one or more uncrosslinked polymers and acrystallizable molecule, (ii) casting the aqueous mixture onto a vessel,a slide, a plate, tissue-culture dish or combinations and modificationsthereof to form a cast mixture, (iii) drying the cast mixture to form anamorphous hydrogel film, (iv) seeding the cast mixture with a seedcrystal of the crystallizable molecule, (v) growing the crystallizablemolecule into a crystal structure within the uncrosslinked polymer, (vi)exposing the cast mixture to ultraviolet light, wherein the exposureresults in a gelling or a crosslinking of the polymer, (vii)crosslinking the uncrosslinked polymer around the crystal structure byan addition of one or more crosslinking agents under conditions in whichthe crystal structure within the crosslinked polymer is maintained,(viii) removing the one or more crystals of the crystallizable polymersby rinsing with water to form the porous hydrogel and (ix) removingwater from the porous hydrogel by controlled dessication under pressure.In one aspect of the method comprises the optional step of surfacecoating, modifying a surface or combinations thereof by soaking thedessicated hydrogel in an aqueous solution comprising the uncrosslinkedpolymer and one or more agents or chemicals to facilitate formation ofone or more bonds. In another aspect the one or more bonds compriseester bonds, amide bonds, carboxylate bonds, carbonyl bonds, etherbonds, imide bonds, and combinations and modifications thereof.

In yet another aspect of the method described hereinabove the polymercomprises nucleic acids, amino acids, saccharides, lipids andcombinations thereof, in monomeric, dimeric, trimeric, oligomeric,multimeric, or polymeric forms. In another aspect the polymer isselected from the group consisting of collagen, chitosan, gelatin,pectins, alginate, hyaluronic acid, heparin and mixtures thereof. In aspecific aspect the polymer comprises a non-synthetic biopolymer that isbiodegradable, biocompatible and hydrophilic. In another aspect thepolymer is gelled by a chemical crosslink, a physical crosslink, or acombination; wherein said crosslink is induced by an UV method, atemperature method, a pH method, an ion, or ion-radical based method orcombinations thereof. In one aspect in the aqueous mixture comprisesalginate and hyaluronic acid. In another aspect the crystallizablemolecule comprises a small organic molecule selected from a salt, urea,beta cyclodextrin, glycine, and guanidine. In a specific aspect thecrystallizable molecule comprises urea.

In one aspect the crosslinking agent selected from group consisting ofcalcium chloride, p-Azidobenzoyl hydrazide,N-5-Azido-2-nitrobenzoyloxsuccinimide, disuccinimidyl glutamate,dimethyl pimelimidate-(2)HCl, dimethyl suberimidate-2 HCl,disuccinimidyl suberate, bis[sulfosuccinimidyl suberate],1-ethyl-3-[3-dimethylaminopropyl]carbodiimide-HCl, isocyanate, aldehyde,glutaraldehyde, paraformaldehyde and derivatives thereof. In anotheraspect the method comprises the optional step of encapsulation one ormore agents selected from drugs, growth factors, hormones, proteins orcombinations thereof in the one or more pores or the matrix of theporous hydrogel.

In another embodiment the instant invention discloses a directionallynetworked porous anti-adhesion hydrogel made by a method that comprisesthe steps of: i) preparing an aqueous mixture of one or moreuncrosslinked polymers and a crystallizable molecule, ii) casting theaqueous mixture onto a vessel, a slide, a plate, tissue-culture dish orcombinations and modifications thereof to form a cast mixture, iii)drying the cast mixture to form an amorphous hydrogel film, iv) seedingthe cast mixture with a seed crystal of the crystallizable molecule, v)growing the crystallizable molecule into a crystal structure within theuncrosslinked polymer, wherein the crystal structure is networked,branched, and porous, vi) exposing the cast mixture to ultravioletlight, wherein the exposure results in a gelling or a crosslinking ofthe polymer, vii) crosslinking the uncrosslinked polymer around thecrystal structure by an addition of one or more crosslinking agentsunder conditions in which the crystal structure within the crosslinkedpolymer is maintained, viii) removing the one or more crystals of thecrystallizable polymers by rinsing with water to form the poroushydrogel, and ix) removing water from the porous hydrogel by controlleddessication under pressure.

The method disclosed hereinabove comprises the optional step of surfacecoating, modifying a surface or combinations thereof by soaking thedessicated hydrogel in an aqueous solution comprising the uncrosslinkedpolymer and one or more agents or chemicals to facilitate formation ofone or more bonds. In one aspect the one or more bonds comprise esterbonds, amide bonds, carboxylate bonds, carbonyl bonds, ether bonds,imide bonds, and combinations and modifications thereof. In anotheraspect the polymer comprises nucleic acids, amino acids, saccharides,lipids and combinations thereof, in monomeric, dimeric, trimeric,oligomeric, multimeric, or polymeric forms. In yet another aspect thepolymer is selected from the group consisting of collagen, chitosan,gelatin, pectins, alginate, hyaluronic acid, heparin and mixturesthereof.

In a related aspect the polymer comprises a non-synthetic polymerbiopolymer, wherein the polymer is biodegradable, biocompatible andhydrophilic. In the method as described above the polymer is gelled by achemical crosslink, a physical crosslink, or a combination; wherein saidcrosslink is induced by an UV method, a temperature method, a pH method,an ion, or ion-radical based method or combinations thereof. In oneaspect the aqueous mixture comprises alginate and hyaluronic acid. Inanother aspect the crystallizable molecule comprises a small organicmolecule selected from a salt, urea, beta cyclodextrin, glycine, andguanidine. In a specific aspect the crystallizable molecule comprisesurea. In another aspect the crosslinking agent selected from groupconsisting of calcium chloride, p-Azidobenzoyl hydrazide,N-5-Azido-2-nitrobenzoyloxsuccinimide, disuccinimidyl glutamate,dimethyl pimelimidate-(2)HCl, dimethyl suberimidate-2 HCl,disuccinimidyl suberate, bis[sulfosuccinimidyl suberate],1-ethyl-3-[3-dimethylaminopropyl]carbodiimide-HCl, isocyanate, aldehyde,glutaraldehyde, paraformaldehyde and derivatives thereof. In anotheraspect the method comprises the optional step of encapsulating one ormore agents selected from drugs, growth factors, hormones, proteins orcombinations thereof in the one or more pores or the matrix of theporous hydrogel. In yet another aspect the hydrogel prevents tissueadhesion following surgery, promotes wound healing, delivers drug orgrowth factors to the support healing, inhibits or prevents infiltrationof blood, blood protein, fibroblasts, and inflammatory responses in thesurgical site and is non-cytotoxic.

In yet another embodiment the present invention relates to a method ofpreventing tissue adhesion during or post-surgery in a patientcomprising the steps of: identifying the patient in need of theprevention of tissue adhesion during or post-surgery amd administeringan injectable solution of an anti-adhesion composition, wherein thecomposition comprises a non-cytotoxic, a non-immunogenic poroushydrogel, a film, a barrier or combinations and modifications thereof,prior to, during or after the surgery, wherein the composition is madeby a method comprising the steps of: a) preparing an aqueous mixture ofone or more uncrosslinked polymers and a crystallizable molecule, b)casting the aqueous mixture onto a vessel, a slide, a plate,tissue-culture dish or combinations and modifications thereof to form acast mixture, c) drying the cast mixture to form an amorphous hydrogelfilm, d) seeding the cast mixture with a seed crystal of thecrystallizable molecule, e) growing the crystallizable molecule into acrystal structure within the uncrosslinked polymer, f) exposing the castmixture to ultraviolet light, wherein the exposure results in a gellingor a crosslinking of the polymer, g) crosslinking the uncrosslinkedpolymer around the crystal structure by an addition of one or morecrosslinking agents under conditions in which the crystal structurewithin the crosslinked polymer is maintained, h) removing the one ormore crystals of the crystallizable polymers by rinsing with water toform the porous hydrogel, and i) removing water from the porous hydrogelby controlled dessication under pressure.

In one aspect the method of making the porous hydrogel comprises theoptional steps of, surface coating, modifying a surface or combinationsthereof by soaking the dessicated hydrogel in an aqueous solutioncomprising the uncrosslinked polymer and one or more agents or chemicalsto facilitate formation of one or more bonds and encapsulating one ormore agents selected from drugs, growth factors, hormones, proteins orcombinations thereof in the one or more pores or the matrix of theporous hydrogel, wherein the hydrogel provides a tunable or a controlledrelease of the one or more agents. In a specific aspect of the methodthe one or more agents comprise ibuprofen or tranexamic acid. In oneaspect the composition the promotes wound healing, delivers drug orgrowth factors to the support healing, inhibits or prevents infiltrationof blood, blood protein, fibroblasts, and inflammatory responses in thesurgical site. In another aspect the polymer is a non-syntheticbiodegradable, biocompatible and hydrophilic biopolymer. In a specificaspect the aqueous mixture comprises alginate and hyaluronic acid. Inanother aspect the crystallizable molecule comprises a small organicmolecule selected from a salt, urea, beta cyclodextrin, glycine, andguanidine. In another aspect the crystallizable molecule comprises urea.

The present invention further discloses a composition for preventingtissue adhesion during or post-surgery in a patient comprising aninjectable solution of an anti-adhesion composition, wherein thecomposition comprises a non-cytotoxic, a non-immunogenic poroushydrogel, a film, a barrier or combinations and modifications thereof.The composition of the present invention is made by a method comprisingthe steps of: i) preparing an aqueous mixture of one or moreuncrosslinked polymers and a crystallizable molecule, ii) casting theaqueous mixture onto a vessel, a slide, a plate, tissue-culture dish orcombinations and modifications thereof to form a cast mixture, iii)drying the cast mixture to form an amorphous hydrogel film, iv) seedingthe cast mixture with a seed crystal of the crystallizable molecule, v)growing the crystallizable molecule into a crystal structure within theuncrosslinked polymer, vi) exposing the cast mixture to ultravioletlight, wherein the exposure results in a gelling or a crosslinking ofthe polymer, vii) crosslinking the uncrosslinked polymer around thecrystal structure by an addition of one or more crosslinking agentsunder conditions in which the crystal structure within the crosslinkedpolymer is maintained, viii) removing the one or more crystals of thecrystallizable polymers by rinsing with water to form the poroushydrogel, and ix) removing water from the porous hydrogel by controlleddessication under pressure. In one aspect the composition isadministered prior to, during or after the surgery

The present invention in one embodiment provides a method of making adirectionally networked porous anti-adhesion hydrogel comprising thesteps of: (i) preparing an aqueous mixture comprising hyaluronic acid,alginic acid, and urea, (ii) casting the aqueous mixture onto a vessel,a slide, a plate, tissue-culture dish or combinations and modificationsthereof to form a cast mixture, (iii) drying the cast mixture to form anamorphous hydrogel film, (iv) seeding the cast mixture with one or moreurea crystals, (v) growing the urea into a crystal structure within theuncrosslinked alginate, (vi) exposing the cast mixture to ultravioletlight, wherein the exposure results in a gelling or a crosslinking ofthe alginate, (vii) removing the one or more urea crystals by rinsingwith water to form the porous hydrogel, and (viii) removing water fromthe porous hydrogel by controlled dessication under pressure.

The method described hereinabove comprises the optional steps of:surface modifying the hydrogel with hyaluronic acid by soaking thedessicated hydrogel in an aqueous solution of the hyaluronic acid in apresence of EDC/NHS and crosslinking the uncrosslinked alginate aroundthe urea crystal structure by an addition of calcium chloride underconditions in which the urea crystal structure within the crosslinkedalginate is maintained. In one aspect the method comprises the optionalstep of encapsulating one or more agents selected from drugs, growthfactors, hormones, proteins or combinations thereof in the one or morepores or the matrix of the porous hydrogel. In another aspect thehydrogel prevents tissue adhesion following surgery, promotes woundhealing, delivers drug or growth factors to the support healing,inhibits or prevents infiltration of blood, blood protein, fibroblasts,and inflammatory responses in the surgical site.

Another embodiment of the instant invention relates to a method formaking a bilayer biofunctionalized HA-based film comprising the stepsof: providing a first layer and a second layer, wherein the first layerand the second layer are made by a method comprising the steps of: (i)preparing an aqueous mixture comprising hyaluronic acid, alginic acid,and urea, (ii) casting the aqueous mixture onto a vessel, a slide, aplate, tissue-culture dish or combinations and modifications thereof toform a cast mixture, (iii) drying the cast mixture to form an amorphoushydrogel film, (iv) seeding the cast mixture with one or more ureacrystals, (v) growing the urea into a crystal structure within theuncrosslinked alginate, (vi) exposing the cast mixture to ultravioletlight, wherein the exposure results in a gelling or a crosslinking ofthe alginate, (vii) removing the one or more urea crystals by rinsingwith water to form the porous hydrogel, and (viii) removing water fromthe porous hydrogel by controlled dessication under pressure and fusingthe first layer and the second layer to form the bilayerbiofunctionalized HA-based film. In one aspect the first layer is adirectionally networked porous anti-adhesion hydrogel. In another aspectthe second layer is a directionally networked porous hydrogel, whereinthe second layer promotes cell adhesion or cell infiltration. In yetanother aspect the method comprises the optional step of covalentlyimmobilizing one or more peptides, proteins, growth hormones or growthfactors on the second layer. In another aspect the peptide is anArginine-Glycine-Aspartic Acid (RGD) peptide. In another aspect thebilayer biofunctionalized HA-based film is used as a nerve wrap, a duralreplacement, a skin graft, for promoting bone in-growth in fracturedressing, chronic wound repair, and patch cardiac or pulmonary tissuesto facilitate tissue repair.

In yet another embodiment the present invention discloses a compositionfor a nerve wrap, a dural replacement, a skin graft, for promoting bonein-growth in fracture dressing, chronic wound repair, patch cardiac orpulmonary tissues to facilitate tissue repair or combinations thereofcomprising a bilayer biofunctionalized HA-based film, wherein the filmis made by a method comprising the steps of: providing a first layer anda second layer, wherein the first layer and the second layer are made bya method comprising the steps of: preparing an aqueous mixturecomprising hyaluronic acid, alginic acid, and urea, casting the aqueousmixture onto a vessel, a slide, a plate, tissue-culture dish orcombinations and modifications thereof to form a cast mixture, dryingthe cast mixture to form an amorphous hydrogel film, seeding the castmixture with one or more urea crystals, growing the urea into a crystalstructure within the uncrosslinked alginate, exposing the cast mixtureto ultraviolet light, wherein the exposure results in a gelling or acrosslinking of the alginate, removing the one or more urea crystals byrinsing with water to form the porous hydrogel, and removing water fromthe porous hydrogel by controlled dessication under pressure and fusingthe first layer and the second layer to form the bilayerbiofunctionalized HA-based film. In one aspect the composition isadministered by an injection, inserted or placed during, after, or priorto a surgical procedure or is applied directly to an affected area.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 is a schematic showing the techniques for fabricating thecrystal-templated biopolymer hydrogels of the present invention;

FIGS. 2A-2D show the surface modification of templated alginate films:FIG. 2A fluorescent biotinylated HA crosslinked to surface labeled withFITC/Neutravadin. When not crosslinked, biotinylated HA washed away(4X), FIG. 2B is a glass slide for FIG. 2A, FIG. 2C is a SEM of thesurface-modified film cross-sectional surface indicating pores filled,scale bar 2 μm, and FIG. 2D is a SEM of a templated film, no surfacemodification, cross-sectional surface indicating unfilled porous, scalebar 1 μm;

FIGS. 3A-3C show Alginate/HA film patterned with an urea crystallizationpattern: FIG. 3A pulling in tension, FIG. 3B crumpling and squeezing,and FIG. 3C returning to original geometry with no tearing or compromiseof integrity;

FIGS. 4A and 4B show the ASTM D638 tensile testing of: FIG. 4A ureapatterned alginate/HA film and FIG. 4B alginate/HA film with nopatterning;

FIGS. 5A and 5B are examples of alginate/HA urea-templated films: FIG.5A linear patterning with 4% urea, 5″ by 5″ film, and FIG. 5B radialpatterning with 6% urea, 3″ by 3″ film;

FIG. 6 is a plot showing the ASTM D638 tensile testing of alginate filmswith increased concentration of urea crystallization;

FIG. 7 is a plot showing the ASTM D638 tensile testing of alginate filmswith increased concentration of HA;

FIGS. 8A and 8B are plots showing the results of the wet sampledegradation studies that were conducted at 37° C. in: FIG. 8A PBS orFIG. 8B 50 IU/mL of hyase. Dashed lines are representative of anestimated degradation since small bits can be seen visually for theduration of the study; and

FIGS. 9A-9E show human dermal fibroblast cells (P=3) were cultured on:FIG. 9A a PLL substrate, FIG. 9B on alginate film, FIG. 9C onalginate/modified HA film or FIG. 9D alginate/modified HA film with HAsurface modification, FIG. 9E is a plot of the % cell adhesion on thedifferent substrates described in FIGS. 9A to 9D.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The instant invention describes a non-toxic anti-adhesion hydrogelbarrier, particularly a barrier composed of non-synthetic, hydrophilic,biodegradable, biocompatible polysaccharides formed by constructing aunique interpenetrating, crosslinked network with a unique porosity, andalso a method for preparing the same. The hydrogel barrier describedherein solves the problems of a film, bulk sponge or nonwoven typeanti-adhesion system, including adhesion to tissue or organs, physicalstrength, in vivo reposition flexibility, ease of handling (i.e.,bending, folding, cutting, rolling, manipulating), and appropriatedegradation timing.

The highly hydrophilic, non-synthetic nature of the barrier of thepresent invention selectively inhibits fibroblast infiltration into asurgical microenvironment and because of its local anti-adhesiveproperties the barrier does not inhibit wound healing. The barrier ofthe present invention does not tear, break or stick to itself whenfolded or rolled and can be easily handled when using surgicalinstruments lending its use in a variety of operations.

The unique features of the present invention are: (i) the barrier iscomprised of tunable biopolymers for controllable mechanical robustnessand degradation, (ii) barrier effectively reduces unwanted adhesionsusing non-synthetic components, and (iii) barrier has unique, controlledhierarchical porosity that can be backfilled with a variety of materialsthat may also be charged with small molecules (drugs, growth factors) tofurther inhibit unwanted response or to support healthy wound healing.No other technology has this combination of features.

The unique benefits provided by the barriers described herein are (i)improved handling characteristics, for example the barrier is easilyfolded, cut, sutured, manipulated in biologically relevant conditions,(ii) persistence in desired area throughout healing duration, (iii)improved in vivo repositioning flexibility, and (iv) unique porousstructure that exhibits a tunable release profile for material, smallmolecules or growth factors.

No other methods in literature are similar to the technique presentedherein. Current anti-adhesion technologies are described herein below

U.S. Pat. No. 6,599,526 discloses a pericardial anti-adhesion patchcomprising a collagenous material and a non-living cellular componentfor preventing adhesion during surgery. U.S. Pat. No. 6,566,345discloses anti-adhesion compositions in the form of a fluid, gel or foammade of intermacromolecular complexes of polysaccharides such ascarboxyl-containing polysaccharides, polyethers, polyacids, polyalkyleneoxides, etc., and synthetic polymers. Korean Patent Publication No.2003-0055102 discloses an anti-adhesion barrier for preventinginflammation and healing wounds comprising carboxymethylcellulose (CMC)and gellan gum. But, the anti-adhesion barriers in the form of a gel,fluid, foam, etc., are not accurately fixed at the wound site; they movedownward because of gravity and, thus, are less effective in healingwounds and reducing adhesion.

European Patent No. 092,733 discloses anti-adhesion barriers in the formof a membrane, gel, fiber, nonwoven, sponge, etc. prepared fromcrosslinking of carboxymethylcellulose (CMC) and polyethylene oxide(PEO). However, carboxymethylcellulose is less biocompatible thanbio-originated materials. Since polyethylene glycol or other syntheticpolymers are not biodegradable, only materials having a small molecularweight that are capable of being metabolized can be used. However, sincematerials having a small molecular weight are absorbed quickly, the roleof the anti-adhesion barrier cannot be sustained sufficiently.

U.S. Pat. No. 6,133,325 discloses membrane type anti-adhesioncompositions made of intermacromolecular complexes of polysaccharidesand polyethers. Korean Patent Publication No. 2002-0027747 disclosesthat a water-soluble polymer gel prepared from alternatingcopolymerization of a block copolymer of p-dioxanone and L-lactide withpolyethylene glycol (PEG) can be utilized as an anti-adhesion barrier,drug carrier, tissue adhesive, alveolar membrane, etc. But, this geltype anti-adhesion barrier is also problematic in accurately fixing atwound sites as the abdominal internal organs or tissues are constantlymoving. U.S. Pat. No. 6,630,167 discloses an anti-adhesion barrierprepared from crosslinked hyaluronic acid. Since hyaluronic acid is apolysaccharide found in animal and human tissues, it has superiorbiocompatibility. However, in an unmodified form, hyaluronic acid isdegraded quickly, with a half life of only 1 to 3 days. This method inparticular claims a crosslinking agent concentration of 10 to 80%, byweight, which is significantly greater than the 1% used in the presentedtechnology. Crosslinking agents can be toxic at high concentrations andremoving large concentrations of crosslinking agents can be difficult.

U.S. Pat. No. 6,693,089 discloses a method of reducing adhesions usingan alginate solution and Korean Patent Publication No. 2002-0032351discloses a semi-IPN (semi-interpenetrating network) type anti-adhesionbarrier using water-soluble alginic acid and CMC, in which alginates areselectively bound to calcium ions. However, these patents includeionically crosslinked alginate by calcium, which, when degraded quickly,releases a bulk charge of calcium ions into the surrounding tissues,further aggravating injured tissues. There is also the problem of nonbio-material uses.

There are publications regarding the treatment of cellulose acetate withsiloxane. But, since celluloses are sensitive to pH, there is adifficulty in processing them. Also, although they are natural polymers,celluloses are not a constituent of the human body and are known to havethe potential to cause a foreign body reaction. Furthermore, thereremains the task of modifying their structure, e.g., through oxidation,so that they can be hydrolyzed inside the body.

Anti-adhesion barriers that are currently on the market are in the formof a film, sponge, fabric, gel, solution, etc. In general, the film orsponge type is easier to fix at a specific site than the solution or geltype. Interceed™ from Johnson & Johnson is the first commercializedanti-adhesion barrier. It is a fabric type product made of ORC andadheres tightly to highly irregular organs or tissues. But, as mentionedearlier, ORC is a non-bio-oriented material and has poorbiocompatibility. Also, because of a very large pore size, cells orblood proteins may easily penetrate the barrier, and the anti-adhesionbarrier is deformed by external force during handling. Seprafilm is afilm type anti-adhesion barrier made of HA and CMC by GenzymeBiosurgery. Seprafilm tends to roll when in contact with water and to bebrittle when dry. Thus, wet hands have to be avoided and moisture shouldbe minimized at the surgical site, which can be very difficult.

HYDROSORB SHIELD® from MacroPore Biosurgery Inc., which is used foradhesion control in certain spinal applications, or SURGI-WRAP™ fromMast Biosurgery, USA which is used after open surgery, are transparentfilm type anti-adhesion barriers made of poly(L-lactide-co-D,L-lactide)(PLA, 70:30), a biodegradable polymer. With a long biodegradation periodof at least 4 weeks and superior mechanical strength, they are known aseasy-to-handle products. Films made of PLA or poly(glycolic acid) (PGA)are easy to roll to one side, but they do not adhere well to thethree-dimensionally, highly irregular surfaces of organs or tissues.Also, since these materials are hydrophobic, they do not absorb moisturewell, and, therefore, do not adhere well to the wet surface of organs ortissues. Also, when hydrolyzed in the body, these materials releaseacidic degradation products, which may cause further inflammation andadhesion. DuraGen® and DuraGen Plus® from Integra LifeSciences is asponge type anti-adhesion barrier made of collagen from an animalsource, which has been developed for surgery and neurosurgery. Since thecollagen sponge absorbs moisture, it readily adheres to the surface oforgans. However, these barriers have relatively weak physical strengthand, because of excessive moisture absorption, tends to be too heavy tohandle or transport to another site.

In general, an anti-adhesion barrier has to satisfy the followingrequirements: i) infiltration or attachment of cells or blood should beavoided through precise control of pore size or use of materialsnon-adherent to blood or cells, ii) the anti-adhesion barrier should beable to be attached at the desired site for a specified period of time,iii) a foreign body reaction should be minimized to reduce inflammation,which is the cause of adhesion, iv) the biodegradation period should beable to be controlled, so that the barrier capacity can be sustained fora requisite period of time, v) the anti-adhesion barrier should beflexible and have superior mechanical properties, including tensilestrength and wet strength, for ease of handling during surgery, and vi)there should be no deformation for a necessary period of time, becausethe wound should be covered exactly.

Post-surgical adhesions tether tissues that should remain separate.Adhesions result from impaired autologous natural immune response.Surgical adhesions continue to plague the recovery period, with currenttechnologies falling short of adhesion prevention. Incidence ofadhesions following surgery is 80% (Yeo, 2007) resulting in chronicpain, limited motion, organ dysfunction, and even death (Cui et al.,2009). The healthcare costs associated with this are over $3.45 billion,annually (Wiseman, et al., 2010). Current approaches for preventingadhesions include better surgical practices (Holmdahl et al., 1997) (fore.g., powder free gloves, laparoscopic procedures, and reduction ofdessication), biocompatible barrier devices (for e.g., polymersolutions, in situ crosslinkable hydrogels, pre-formed membranes), andpharmacotherapy agents like steroidal anti-inflammatory drugs(Dexamethasone; progesterone; hydrocortisone; prednisone), non-steroidalanti-inflammatory drugs (Ibuprofen; flurbiprofen; indomethacin;tolmetin; nimesulide), inhibitors of proinflammatory cytokines(Antibodies to transforming growth factor (TGF)-b1), antihistamine(Diphenhydramine; promethazine), free radical scavengers (Melatonin;vitamin E; superoxide dismutase), Anticoagulants (heparin), proteolyticagents (tissue-type plasminogen activator; streptokinase; urokinase;pepsin; trypsin; Neurokinin 1 receptor antagonist), andantiproliferative agents (mitomycin).

The most effective anti-adhesion barrier on the market reduces adhesionformation by only 50%. Many products are based on synthetic materialsbecause of superior handling capabilities and low manufacturing costs.However, these synthetic materials are rendered ineffective in thepresence of blood or blood proteins. The invention presented hereinaddresses the problems listed above and provides an effective method ofblocking the infiltration of unwanted inflammatory response whilemaintaining robust mechanical properties for surgical handling. Becausethe present invention is constructed of natural materials, the risk offurther aggravation is minimized, while blood and blood proteins willnot adhere. Barriers on the market made from natural materials alsodegrade too quickly, allowing for adhesion formation. The presenttechnology has a tunable degradation rate so that the barrier persistsduring the healing process.

Current products on the market that are most effective have poorhandling properties. They are brittle when dry and are renderedinapplicable when wet. In an OR environment, a suitable solution wouldbe able to maintain mechanical integrity when wet. The present inventionoffers superior handling properties when wet including in vivorepositioning capabilities and suturability.

The present invention describes the development of composite,dual-functioning materials to be placed at the interface between healingtissues and the surrounding tissues. The invention improves uponanti-adhesive biomaterial barriers, to aid in wound healing, and tomodulate the inflammatory response. The present inventors have developand characterize anti-adhesive HA-based material (biocompatible,non-immunogenic, non cell-adhesive, inhibits protein absorption,mechanically stable, cost effective, clinically sized, and appropriatedegradation rate). In addition the present inventors have developed abilayer biofunctionalized HA-based film that is biocompatible,bioabsorbable, non-immunogenic, dual functioning, regenerative,anti-adhesive, mechanically stable, cost effective, and clinicallysized. Finally, they develop an injectable solution version ofanti-adhesive film that is biocompatible, effective at reducingadhesions, encapsulates ibuprofen or tranexamic acid and has tunablerelease rates.

Hydrogels are generally polymer chain networks that are water-insoluble,but that absorb water. Often described as being “superabsorbent,”hydrogels are able to retain up to 99% water and can be made fromnatural or synthetic polymers. Often, hydrogels will have a high degreeof flexibility due to their high water content. Common uses forhydrogels include: sustained drug release, as scaffolds (e.g., in tissueengineering), as a thickening agent, as a biocompatible polymer, inbiosensors and electrodes and for tissue replacement applications.Natural hydrogels may be made from agarose, methylcellulose, hyaluronicacid (HA), and other naturally-derived polymers.

HA is a linear polysaccharide with repeating disaccharide units composedof sodium D-glucuronate and N-acetyl-D-glucosamine. This naturallyoccurring glycosaminoglycan is a component of skin, synovial fluid, andsubcutaneous and interstitial tissues. HA is metabolically eliminatedfrom the body, and plays a role in protecting and lubricating cells andmaintaining the structural integrity of tissues. Anionic carboxylicgroups immobilize water molecules giving HA its viscoelastic and anticell-adhesive properties. HA has been used in a variety of materialdesigns for the prevention of postsurgical tissue adhesion. HA has beenused as a dilute solution, a crosslinked hydrogel, or combined with CMCinto sheets. HA is biocompatible, bioabsorbable/non-immunogenic(non-animal), very non-cell adhesive, polyanionic, hydrophilic,antifibrotic (1% HMW HA, Massie, 2005), pro-angiogenic and has beenshown to reduce adhesion formation in animals and humans (Zawaneh, 2008;Diamond, 2006; Wiseman, 2010; Rajab, 2010). HA is clinically used toreduce adhesions: Seprafilm®, most effective and widely usedanti-adhesion barrier on the market.

Alginic acid is biocompatible, bioabsorbable/non-immunogenic(non-animal) (Skjak-Braek, 1992), very non-cell adhesive, polyanionic,hydrophilic, cost effective, abundant (brown seaweed), mechanicallyviable for handling/suturing in ionically crosslinked form, and is shownto be significantly effective at adhesion prevention in animal models(Namba, 2006; Cho, 2010a; Cho, 2010b).

Attributes of alginate that statistically alter mechanical properties:(i) grade (Purification), (ii) gulcuronate to mannuronate ratio (High Mratio is pond-grown, primarily leaves, High G is deep sea harvested,primarily stems), and (iii) molecular weight/viscosity. However, highlypurified alginate is very expensive˜$100/g, lower grade (inexpensive)alginates are not tested for molecular weight or G:M ratio, andpurification processes are not standardized.

Crystal templated hydrogels of alginate and HA were created by casting adroplet of solution containing a photocrosslinkable derivative of HA, aphotocrosslinkable derivative of alginate with photoinitiator (PI) andurea (FIG. 1). The solvent is evaporated and a urea seed crystal istouched to the droplet to nucleate urea crystallization. Aftercrystallization the alginate and HA are photocrosslinked by UV exposure.Alginate may be further crosslinked ionically and rinsed with water toremove the urea leaving behind an alginate/HA hydrogel templated withthe pattern of the urea crystals. The hydrogel may then be dehydratedfor further surface modification using crosslinking agents (such aswater soluble carbodiimides in ethanol/deionized water mixtures).

The method for preparing the alginate/HA films as described in thepresent invention includes five steps: film casting, solventevaporation, crystal growth, crosslinking, and rinsing. In the firststep a syringe filter introduces a solution comprisingalginate/GMHA/urea on a plate. The solution is then cast as a film at25° C. at 70% relative humidity. Solvent evaporation is required toachieve the super-saturation conditions necessary for crystallization.Evaporation also greatly increases the biopolymer concentration andsolution viscosity. The combination of high viscosity and hydrogenbonding suppresses spontaneous urea crystallization and facilitatessuper-saturation. Urea seed crystals are deposited on the tips of a finepair of tweezers and is added to nucleate crystallization followed byexposure to UVA (500 mW/cm²) for 15 secs. Crystal growth beganimmediately and produced long dendritic branches that extended from thecenter to the edge of the film. Within seconds the entire volume of thehydrogel films were filled with urea crystals. These crystals comprisedthe urea crystal template. The films may optionally be crosslinked by anaddition of one or more cross linking agents (for example an ioniccrosslinking solution like CaCl₂ is added to the film to crosslink thealginate). The urea crystals are then rinsed out with double distilledwater. The film formed thus is subjected to controlled dessication underforce to remove water at 50% relative humidity. The dehydrated film maybe subjected to further surface modification by creating one or moreester or less hydrolysable bonds by a variety of techniques (got e.g.,soaking in a HA solution using water soluble carbodiimide for esterbonds).

Alginate films alone degraded too quickly in chelating environment.Calcium ions chelated by multiple salts and can degrade within a fewhours. (Islam, 2010). Adding GMHA decreases degradation, but withoutcompromising the mechanical strength provided by alginate. Alginatefilm, alone, is too brittle and breaks with little manipulation. Addingurea introduces micron-sized pores which provide flexibility becausespaces accept forces first.

FIGS. 2A-2D show the surface modification of templated alginate films.FIG. 2A shows fluorescent biotinylated HA crosslinked to surface labeledwith FITC/Neutravadin. When not crosslinked, biotinylated HA washed away(4X). FIG. 2B is a glass slide for FIG. 2A. FIG. 2C are is a SEM of thesurface-modified film cross-sectional surface indicating pores filled,scale bar 2 μm and of a templated film, no surface modification,cross-sectional surface indicating unfilled porous, scale bar 1respectively.

FIGS. 3A-3C are images showing the integrity of the Alginate/HA filmpatterned with an urea crystallization pattern, pulling in tension (FIG.3A), crumpling and squeezing (FIG. 3B), and returning to originalgeometry with no tearing or compromise of integrity (FIG. 3C).

The ASTM D638 tensile testing of urea patterned alginate/HA film andalginate/HA film with no patterning is shown in FIGS. 4A and 4B. Thepatterned film recoils in response to plastic deformation beforefailure. The non-patterned film breaks with a brittle fracture. Examplesof alginate/HA urea-templated films are shown in FIGS. 5A and 5B, linearpatterning with 4% urea, 5″ by 5″ film (5A), and radial patterning with6% urea, 3″ by 3″ film (5B).

FIG. 6 is a plot showing the ASTM D638 tensile testing of alginate filmswith increased concentration of urea crystallization. The trendindicates increased plasticity with increased crystallizationpatterning. FIG. 7 is a plot showing the ASTM D638 tensile testing ofalginate films with increased concentration of HA. The trend indicatesdecreased tensile strength with increased HA, until a critical point,where the concentration of HA improves the tensile strength by providingcrosslinked strength.

Characterization of synthesized Alginate/HA films: FIGS. 8A and 8B areplots showing the results of the wet sample degradation studies of anAlginate/HA film of the present invention conducted at 37° C. in PBS or50 IU/mL of hyase, respectively. Briefly, the method involvesdetermining a pre-test weight of the Alginate/HA film (or films)(W_(o)), after that the film is placed in PBS or the hyase at 37° C. andare removed at pre-defined time points and weighed (W_(f)). Theprocedure is repeated until the weight cannot be taken or theappropriate pre-defined end point time is reached. The degradation rateis calculated using the formula given below:% Weight Loss=100×(W _(o) −W _(f))/W _(o)

Dashed lines are representative of an estimated degradation since smallbits can be seen visually for the duration of the study. Alginate alonefilms degrade due to chelating agents in the buffer.

FIG. 9 is a schematic showing the steps involved in the evaluation ofthe anti-cell adhesion properties of an alginate/HA film of the presentinvention. A culture of fibroblast cells is taken, half of it isretained on a TCPS dish and the other half is retained on an Alginate/HAfilm that has been placed on a dish. After a 24 hour waiting period,both the dishes are stained with calcein/ethidium to label the live ordead cells. The cell adhesion or non cell adhesion is validated usingone or more commonly used cell technologies.

To study cell adhesion properties human dermal fibroblasts cells (P=3)were cultured on a PLL substrate, on alginate film, on alginate/modifiedHA film or alginate/modified HA film with HA surface modificationalginate/HA film of the present invention (FIGS. 9A-9D). A culture offibroblast cells is taken and split in half and placed separately on twoTCPS dishes. The film/substrate to be tested is placed on top of one ofthe dishes. After a 6 hour waiting period, both the dishes are stainedwith calcein/ethidium to label the live or dead cells. FIG. 9E is a plotof the % cell adhesion on the different substrates described in FIGS. 9Ato 9D. Leaching studies showed no cytotoxic results from film, stainingat 24 hours.

The barrier disclosed hereinabove possesses significant advantages overcurrently existing technologies: (1) the barrier has improved handlingcharacteristics, is easily folded, cut, sutured, manipulated inbiologically relevant conditions; (2) barrier persists in desired areathroughout healing duration; (3) barrier has improved in vivorepositioning flexibility; and (4) unique porous structure that exhibitsa tunable release profile for material, small molecules, or growthfactors.

The unique anti-adhesive membrane described hereinabove could also be aninnovative solution in the enormous wound care market. As a substratefor a non-adhesive, hydrophilic, yet absorbent wound dressing, thepresent invention can be used extensively in burn care, chronicnon-healing wound care, and reconstructive plastic surgery.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A method comprising: preparing a first aqueousmixture comprising hyaluronic acid and an alginate, casting the firstaqueous mixture onto a first medium to form a first cast mixture, dryingthe first cast mixture to form a first hydrogel film, crosslinking thealginate of the first hydrogel film, and removing water from the firsthydrogel film by desiccation under pressure; preparing a second aqueousmixture comprising hyaluronic acid and an alginate, casting the secondaqueous mixture onto a second medium to form a second cast mixture,drying the second cast mixture to form a second hydrogel film,crosslinking the alginate of the second hydrogel film, and removingwater from the second hydrogel film by desiccation under pressure;fusing the first hydrogel film with the second hydrogel film to form amulti-layer film; wherein the multi-layer film is at least partiallydesiccated.
 2. The method of claim 1, wherein the multi-layer film isamorphous and translucent.
 3. The method of claim 1, comprisingcrosslinking the alginate of the first hydrogel film with at least onecation.
 4. The method of claim 3, comprising crosslinking the alginateof the second hydrogel film with the at least one cation.
 5. The methodof claim 4, wherein the at least one cation includes calcium.
 6. Themethod of claim 3 wherein the first hydrogel film includes uncrosslinkedhyaluronic acid.
 7. The method of claim 6, wherein at least one of thefirst and second hydrogel films is anti-adhesive and non-attractive tocells.
 8. The method of claim 7, wherein both of first and secondhydrogel films are anti-adhesive and non-attractive to cells.
 9. Themethod of claim 6, wherein: the first hydrogel film is anti-adhesive andnon-attractive to cells; and the second hydrogel film is adhesive tocells.
 10. The method of claim 1, wherein the first hydrogel film is noless than 1% hyaluronic acid by dry weight and no greater than 33%hyaluronic acid by dry weight.
 11. The method of claim 10, wherein: thehyaluronic acid and the alginate in the first hydrogel film are formedaround a pore network; the method comprises fully crosslinking thealginate of the first hydrogel film and fully crosslinking the alginateof the second hydrogel film.
 12. A method comprising: preparing firstand second aqueous mixture portions each comprising hyaluronic acid andan alginate; casting the first and second aqueous mixture portions toform first and second cast mixture portions; removing water from thefirst and second hydrogel film portions by desiccation under pressure;drying the first and second cast mixture portions to form first andsecond hydrogel film portions; crosslinking the alginate of the firstand second hydrogel film portions; and fusing the first hydrogel portiononto the second hydrogel film portion to form a multi-layer film;wherein the multi-layer film is at least partially desiccated.
 13. Themethod of claim 12, comprising crosslinking the alginate of the firsthydrogel film portion with at least one cation.
 14. The method of claim13, comprising crosslinking the alginate of the second hydrogel filmportion with the at least one cation.
 15. The method of claim 13,wherein at least one of the first and second hydrogel film portions isanti-adhesive and non-attractive to cells.
 16. The method of claim 15,wherein both of first and second hydrogel film portions areanti-adhesive and non-attractive to cells.
 17. The method of claim 13,wherein: the first hydrogel film portion is anti-adhesive andnon-attractive to cells; and the second hydrogel film portion isadhesive to cells.
 18. The method of claim 13, wherein: the hyaluronicacid and the alginate in the first hydrogel film portion are formedaround a pore network; the method comprises fully crosslinking thealginate of the first and second hydrogel film portions.